MINIREVIEW Epigenetics: the study of embryonic stem cells by restriction landmark genomic scanning Naka Hattori* and Kunio Shiota Laboratory of Cellular Biochemistry, Animal Resource Sciences ⁄ Veterinary Medical Sciences, University of Tokyo, Japan Differentiation of a specific cell type involves the establishment of a precise epigenetic profile comprised of genome-wide epigenetic modifications such as DNA methylation and histone modification. Because epi- genetic modifications in gene areas regulate transcrip- tional activity, the epigenetic profile of the cell reflects the transcriptome of the cell, at least partially. DNA methylation is a major component of epigenetic modi- fication in mammals [1,2]. The DNA methylation pro- file at tissue-specific differentially methylated regions (originally named tissue-dependent and differentially methylated regions: T-DMRs) in one cell type is differ- ent from others and represents a unique property of the cell [3,4]. However, the precise mechanism behind formation of the epigenetic profile, including the DNA methylation profile during development, remains to be elucidated. A wide range of methods has been developed for qualitative and quantitative DNA methylation assays [5]. Although methods based on microarray technology are undoubtedly useful and promising for analyzing whole-genome profiles of DNA methylation, as well as histone modifications [4], restriction landmark genomic scanning (RLGS), which is based on 2D electrophore- sis in combination with methylation-sensitive restric- tion enzymes [6], is still a powerful method for DNA Keywords DNA methylation; DNA methylation profile; Dnmt; epigenetics; ES cells; histone methylase; histone modification; mammalian development; RLGS; T-DMR Correspondence N. Hattori, Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan Fax: +81 44 244 9617 Tel: +81 44 210 5959 E-mail: naka_hattori@ajinomoto.com *Present address Institute of Life Sciences, Ajinomoto Co., Inc., Japan (Received 30 November 2007, revised 25 January 2008, accepted 29 January 2008) doi:10.1111/j.1742-4658.2008.06331.x During mammalian development, it is essential that the proper epigenetic state is established across the entire genome in each differentiated cell. To date, little is known about the mechanism for establishing epigenetic modi- fications of individual genes during the course of cellular differentiation. Genome-wide DNA methylation analysis of embryonic stem cells by restriction landmark genomic scanning provides information about cell type- and tissue-specific DNA methylation profiles at tissue-specific methy- lated regions associated with developmental processes. It also sheds light on DNA methylation alterations following fetal exposure to chemical agents. In addition, analysis of embryonic stem cells deficient in epigenetic regulators will contribute to revealing the mechanism for establishing DNA methylation profiles and the interplay between DNA methylation and other epigenetic modifications. Abbreviations Dnmt, DNA methyltransferase; EB, embryoid body; ED, epigenetic distance; EG cell, embryonic germ cell; ES cell, embryonic stem cell; RLGS, restriction landmark genomic scanning; T-DMR, tissue-specific differentially methylated region or tissue-dependent and differentially methylated region; TS cell, trophoblast stem cell; Vi-RLGS, virtual image restriction landmark genomic scanning. 1624 FEBS Journal 275 (2008) 1624–1630 ª 2008 The Authors Journal compilation ª 2008 FEBS methylation analysis. Although RLGS requires a larger genomic sample than is necessary for microarray-based methods, it has advantages for analyzing genome-wide methylation states: (a) it is a highly reproducible quan- titative method; (b) genomic DNA is not amplified, thus limiting or avoiding detection bias; (c) it detects unmethylated landmarks in the genome and keeps out repeated sequences that are usually highly methylated; and (d) it targets predominantly CpG islands by using restriction enzymes that have recognition sites with high CG contents, such as NotI. Moreover, virtual image RLGS (Vi-RLGS), a recently developed soft- ware simulating RLGS in silico using genomic sequences, overcomes the difficulty in identifying sequences of RLGS fragments [7]. One of the most important advances in develop- mental biology and cell biology is the establishment of embryonic stem (ES) cells, which maintain the ability to form all types of cells in the body, and can differentiate into a variety of cell types in vitro [8]. The use of ES cells in epigenetic studies enables us to analyze how epigenetic profiles change during devel- opmental processes and the effects on epigenetic regulators of fetal exposure to chemical agents. In addition, gene targeting of epigenetic regulators in ES cells allows us to investigate the role of each epi- genetic regulator in establishing the epigenetic profile, and study the interplay between epigenetic modifica- tions such as DNA methylation and histone modifica- tion. In this minireview, we describe studies using RLGS to analyze DNA methylation profiles in ES cells. Investigation of DNA methylation profiles during mammalian development using ES cells In the mammalian genome, DNA methylation occurs in T-DMRs according to cell- or tissue-type to regulate the expression of neighboring genes [3]. By comparing 10 different cell types and tissues, we previously revealed that 247 T-DMRs existed among 1500 genomic loci, and that DNA methylation pro- files comprise the methylation status of the T-DMRs [9]. The DNA methylation profile of 247 T-DMRs was identified as a unique code for the cell or tissue [3,4]. Considering that there are more than 15 000 CpG islands in the mouse haploid genome, of which RLGS can only sample a subset, and that there are  200 cell types in mammals, the number of identi- fied T-DMRs is likely to expand in future studies, exposing even more complex DNA methylation profiles. Differences in DNA methylation profiles between ES and other stem cells Comparing ES cells with other stem cells established from developing embryos revealed the uniqueness of the epigenetic profile in ES cells. In contrast to ES cells, which maintain the ability to differentiate into all cell types of the embryo proper [10], trophoblast stem (TS) cells originate from the trophectoderm of blast- ocysts and can differentiate only into placental cells in vivo and in vitro [11]. Differentiation of cells from the early blastomere stage to the blastocyst stage is accompanied by a change in the epigenetic profile that directs the differentiation pathway to either the embryo proper or the placenta. Thus, a significant dif- ference between ES and TS cells is likely to be observed by comparing their epigenetic profiles. Analy- sis by RLGS revealed that DNA methylation profiles at T-DMRs are totally different between ES and TS cells [9]. Compared with TS cells, 20 genomic loci were methylated and 57 loci were demethylated in ES cells, supporting the idea that a bifurcation of the epigenetic profile exists before development of the blastocyst. Embryonic germ (EG) cells are known to have simi- lar characteristics to ES cells with respect to differenti- ation and proliferation capabilities, despite their different origins [12,13]. It was demonstrated that glo- bal gene-expression profiles of ES and EG cells were indistinguishable [14]. However, analysis of DNA- methylation profiles by RLGS revealed a significant difference between ES and EG cells [9]. Among 1500 genomic loci in the RLGS profile, 49 (3%) were found to be methylated differentially in ES and EG cells, indicating that ES and EG cells can be distinguished from each other by the DNA methylation profiles. If we defined ‘epigenetic distance’ (ED) as the number of differentially methylated loci per 1500 genomic loci of two given cell- or tissue types, the ED between ES and EG cells (49) is less than that between ES and TS cells (77), confirming the previous notion that EG cells are more similar to ES cells than to TS cells (Fig. 1). Change of DNA methylation profiles during the developmental process To examine how the DNA methylation profile changes as the embryo develops, we utilized model differentia- tion systems and analyzed the DNA methylation pro- files of ES cells, embryoid bodies (EBs), teratomas derived from the same ES cells, fetuses at E10.5 and adult organs [15]. Teratomas are disorganized agglom- erates with tissue or organ components derived from all three germ layers. Teratomas, as well as fetuses, have N. Hattori and K. Shiota Epigenetic study of embryonic stem cells FEBS Journal 275 (2008) 1624–1630 ª 2008 The Authors Journal compilation ª 2008 FEBS 1625 DNA methylation profiles that are obtained from a mixture of heterogeneous tissues or organs, meaning that the methylation status at each locus in a profile reflects average levels of DNA methylation of all cell types analyzed. Thus, detectable alterations in the DNA methylation profiles of teratomas or embryos indicate common alterations that occurred in the whole teratoma or embryo concurrent with the differentiation of ES cells. Among the 259 T-DMRs, including the ori- ginal 247 T-DMRs [9], the fraction of methylated loci, which was 51.4% in ES cells, was lower in fetuses (40.2%) and brain of adult mice (48.6%) but higher in kidney (53.7%). A similar change was observed in the in vitro differentiation system; methylation levels were low (39.6%) in EBs and higher (41.3–44.4%) in three independently developed teratomas derived from ES cells or EBs. The number of methylated loci in the profiles of teratomas was less than that of the somatic tissues, probably because the teratomas still contained a significant number of undifferentiated proliferating cells, or all cells in teratomas were not fully differenti- ated yet. Because the methylation status of T-DMRs partially corresponds with the transcriptional status of the neighboring gene, identifying differentially methyl- ated genomic loci in ES cells, EBs and teratomas is expected to provide information about genes that are responsible for the developmental process. Potential of ES cells in embryotoxicological studies Embryonic exposure to chemical agents or medicine may have deleterious effects on proper embryogenesis, especially during the early developmental stages. Such agents may influence embryos at genetic, transcriptional and protein levels. It is also conceivable that epigenetic alterations occur with exposure of embryos to these agents, because epigenetic profiles are established actively in developing embryos. Differentiation of ES cells into EBs has been studied as an in vitro model of normal and abnormal mammalian development [16]. Because differentiation from ES cells to EBs is accom- panied by changes in DNA methylation profiles at T-DMRs [15], the in vitro differentiation model is useful to assess the epigenetic effect of an agent on the developmental process, and helps avoid the ethical issue of embryotoxicological surveillance of ‘epimutagens’ [17]. In addition, it is necessary to assess the effects of agents on the ES cell itself, for future therapeutic use in regenerative medicine. For example, dimethyl sulfoxide, an amphipathic molecule, is a commonly used cryopre- servative for various cells, including ES cells, and a sol- vent for water-insoluble substances in cytological and cytotoxicological studies [18]. It has been reported that exposure to dimethyl sulfoxide induced differentiation in several types of cells [18], and that dimethyl sulfoxide could improve the frequency of development to the blastocyst stage after nuclear injection in mouse cloning [19]. RLGS analysis revealed that dimethyl sulfoxide treatment of ES cells differentiating into EBs, at con- centrations lower than when used as a cryopreservative, resulted in the alteration in the DNA methylation profile [20]. Both hypo- and hypermethylation were observed at T-DMRs depending on the genomic loci, with hypermethylation occurring at minor satellite repeats and endogenous C-type retroviruses. Among epigenetic regulators, including DNA methyltransferas- es (Dnmts) and histone modification enzymes, Dnmt3a subtypes were upregulated both at the mRNA and pro- tein level in dimethyl sulfoxide-treated cells, suggesting that dimethyl sulfoxide might have a direct impact on DNA methylation via up-regulation of Dnmt3a sub- types, at least, at hypermethylated loci and repetitive sequences. Analysis of the DNA methylation profile for therapeutic use of human ES cells in regenerative medicine The potential use of human ES cells in the field of regenerative medicine has been discussed previously, and differentiation of human ES cells into various tis- sues has been investigated [8]. Several lines of human ES cells were established, and differences between these ES cell lines with respect to karyotypic stability [21] and expression profiles [22] have been investigated. It ICM TE PGC TS cells ES cells EG cells 4977 Placental cells Embr y onic cells Fig. 1. Epigenetic distances between ES cells and other stem cells derived from developing embryos. ES cells derived from the inner cell mass (ICM) of blastocysts and EG cells derived from the pri- mordial germ cells (PGCs) in developing genital ridges can develop into cells of the embryo proper, after they are injected into blast- ocysts to form chimeras. By contrast, TS cells derived from the trophectoderm (TE) of blastocysts contribute only to placenta. Although there is an apparent ED between ES cells and EG cells, the ED of TS cells to ES cells (77) is greater than that of EG cells to ES cells (49), confirming the similarity of EG cells to ES cells. Epigenetic study of embryonic stem cells N. Hattori and K. Shiota 1626 FEBS Journal 275 (2008) 1624–1630 ª 2008 The Authors Journal compilation ª 2008 FEBS has been demonstrated that mouse and human ES cells have unique DNA methylation profiles compared with other cell types, including EG cells, TS cells and sev- eral adult stem cell populations [9,23]. Also, key regu- lators of development such as Oct-4 and Nanog are controlled by epigenetic mechanisms [24,25]. To ensure the safe use of ES cells for regenerative medicine, it will be necessary to evaluate the nature of differenti- ated cells as thoroughly as possible. Accordingly, it is also important to evaluate the epigenetic stability of ES cell lines. Using RLGS, Allegrucci and co-workers investigated the DNA methylation profiles of indepen- dently isolated human ES cells after culture under vari- ous conditions [26]. They demonstrated that variations in DNA methylation profile existed between ES cell lines, which could not be accounted for by genetic dif- ferences of the source embryos. Although the number of cell passages and culture conditions, such as the existence of serum or feeder-layer, affected neither morphology nor expression of cell markers, these parameters changed the DNA methylation profile of human ES cells. Considerable numbers of loci with different DNA methylation status were also aberrantly methylated in human tumor cells [27]. Investigation of epigenetic mechanisms with ES cells deficient in epigenetic regulators Homologous recombination in ES cells enables us to perform gene targeting at specific chromosomal loci and to investigate gene function [28]. In addition, knockout mice have been generated to study the devel- opmental role of the gene by germline transmission of a targeted allele. Genetic manipulations of many epige- netic regulators, including Dnmts [29–33] and histone methylases [34,35], have been reported. Genome-wide DNA methylation analysis of ES cells deficient in epi- genetic regulators will assist in revealing the mecha- nism for maintaining DNA methylation in T-DMRs, as well as the interplay between DNA methylation and other epigenetic modifications. Mechanism for maintaining DNA methylation at T-DMRs Based on studies regarding the properties of Dnmts, it is widely accepted that Dnmt1 is a maintenance DNA methyltransferase and Dnmt3a ⁄ 3b are de novo DNA methyltransferases in vivo [36]. Dnmt3a and Dnmt3b have no preference for hemimethylated DNA [37], and a transgene of Dnmt3a, but not of Dnmt1, to Drosophila exhibited de novo methylation activity [38], indicating that Dnmt3a ⁄ 3b function in de novo DNA methylation, but not in maintenance DNA methylation. However, following these studies, it was still unclear how Dnmt1 and Dnmt3a ⁄ 3b are involved in DNA methylation in T-DMRs, thereby establishing DNA methylation profiles of cells, and whether Dnmt3a ⁄ 3b have any role in maintenance DNA methylation in T-DMRs. We demonstrated cooperation of Dnmt1 and either Dnmt3a or Dnmt3b in the maintenance of DNA meth- ylation in gene areas [39]. Using RLGS with Dnmt1-, Dnmt3a- and ⁄ or Dnmt3b-deficient ES cells, we focused on the involvement of Dnmts in the methylation of CpG islands and CpG-rich regions near genes. Both Dnmt1 single mutation and Dnmt3a ⁄ Dnmt3b double mutation in ES cells resulted in the demethylation of many loci. Surprisingly, target T-DMRs of Dnmt1 were identical to those of Dnmt3a ⁄ Dnmt3b. Although a single disruption of Dnmt3a or Dnmt3b resulted in no change in DNA methylation at the same loci, it was shown that maintaining DNA methylation at identified loci requires both classes of Dnmts, Dnmt1 and either Dnmt3a or Dnmt3b. Kinetic analysis of ES cells defi- cient in Dnmts indicated that demethylation in repeat sequences was progressive in Dnmt3a ⁄ 3b-deficient ES cells, with notable demethylation during later stages of cell culture, whereas demethylation in Dnmt1-deficient ES cells was more rapid and greater during the initial stages of culture [40]. This implies a predominant role for Dnmt1 and supportive role for Dnmt3a and Dnmt3b in maintaining DNA methylation at the repeat sequences. By contrast, further analysis by bisulfite sequencing of loci studied by RLGS determined that extensive and almost complete demethylation occurred at genes in Dnmt3a ⁄ 3b-deficient ES cells, whereas demethylation was rather moderate in Dnmt1-deficient ES cells [39]. It is probable that in Dnmt1-deficient ES cells, Dnmt3a and Dnmt3b exert de novo DNA methyl- ation activity at these genes, which are demethylated through lack of maintenance activity because Dnmt1 is absent. Consequently, Dnmt1-deficient ES cells seem to have partial DNA methylation maintenance activity, which is provided by the re-methylating actions of Dnmt3a ⁄ Dnmt3b (Fig. 2). Dnmt3a and Dnmt3b appear to function both as maintenance and as de novo methyltransferases in gene areas, and thus are crucial for the establishment of the DNA methylation profile during development. Analyzing the interplay between DNA methylation and histone methylation Chromatin structure, which is affected by DNA meth- ylation and histone modification, is closely associated N. Hattori and K. Shiota Epigenetic study of embryonic stem cells FEBS Journal 275 (2008) 1624–1630 ª 2008 The Authors Journal compilation ª 2008 FEBS 1627 with the transcriptional activity of genes. During mam- malian development, the epigenetic profile is not estab- lished solely by one particular epigenetic regulator, but rather by the interplay of epigenetic regulators [41,42]. The relationship between DNA methylation and other epigenetic modifications can be examined by genome- wide DNA methylation analysis using ES cells defi- cient in epigenetic regulators. Growing evidence has indicated that histone lysine methylation can direct DNA methylation in many organisms [43]. G9a is a euchromatin-localized histone methylase that catalyzes the methylation of histone H3 at Lys9 and Lys27 (H3–K9 and H3–K27) [44], which are often found in heterochromatic regions and in transcriptionally inac- tive loci of the genome [45]. RLGS analysis of G9a- deficient ES cells revealed a direct interaction between DNA methylation and H3–K9 and H3–K27 methyla- tion at T-DMRs during ES cell differentiation [46]. In G9a-deficient ES cells, the levels of DNA methylation decreased in some genomic loci, and Vi-RLGS revealed the location of these loci in euchromatic regions. Chromatin-immunoprecipitation confirmed the demethylation of H3–K9 and H3–K27 at genomic loci following G9a knockout, indicating that demethyl- ation of H3–K9 and H3–K27 triggered the disruption of maintenance DNA methylation. Restoration of G9a activity by insertion of the transgene into G9a-deficient ES cells resulted in full recovery of methylation levels to almost all genomic loci. This suggests that G9a also facilitates de novo DNA methylation of the target loci. Because G9a does not have the cata- lytic domain of Dnmts, G9a plays a role in DNA methylation indirectly, possibly via methylation at H3–K9 and ⁄ or H3–K27. This study also suggests the potential to discover novel targets of an epigenetic regulator that affects DNA methylation, by analyzing alterations in DNA methylation in cells deficient in the factor. Conclusions Genome-wide DNA methylation analysis of ES cells has the potential to reveal the mechanisms used to establish DNA methylation profiles, and the epigenetic effects of fetal exposure to chemical agents during mammalian development. An increased number of ES cell lines deficient in epigenetic regulators will facilitate investigations into the interplay between DNA methyl- ation and other epigenetic modifications through identification of DNA methylation profiles by RLGS or other genome-wide analysis methods. Acknowledgements We thank M. Higgins for reviewing the original manu- script. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). References 1 Bird AP & Wolffe AP (1999) Methylation-induced repression – belts, braces, and chromatin. Cell 99, 451– 454. 2 Bird A (2002) DNA methylation patterns and epigenetic memory. 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MINIREVIEW Epigenetics: the study of embryonic stem cells by restriction landmark genomic scanning Naka Hattori* and Kunio Shiota Laboratory of Cellular. other stem cells Comparing ES cells with other stem cells established from developing embryos revealed the uniqueness of the epigenetic profile in ES cells.